The final step in the blood coagulation cascade is the thrombin-catalyzed conversion of the soluble plasma protein fibrinogen to insoluble fibrin. Thrombin cleaves a small peptide (fibrinopeptide A) from one of the three component chains (the Aα-chain) of fibrinogen. Fibrin monomers subsequently polymerize and are cross-linked by activated factor XIII to form a stable clot.
Fibrinogen is a key component of biological tissue glues (see, e.g., U.S. Pat. Nos. 4,377,572 and 4,442,655), which mimic the formation of natural blood clots to promote hemostasis and repair damaged tissue. Tissue glues provide an adjunct or alternative to sutures, staples and other mechanical means for wound closure. However, the principal ingredients of these products (fibrinogen, factor XIII and thrombin) are prepared from pooled human plasma by cryoprecipitation (e.g. U.S. Pat. Nos. 4,377,572; 4,362,567; 4,909,251) or ethanol precipitation (e.g. U.S. Pat. No. 4,442,655) or from single donor plasma (e.g. U.S. Pat. No. 4,627,879; Spotnitz et al., Am. Surg. 55: 166-168, 1989). The resultant fibrinogen/factor XIII preparation is mixed with bovine thrombin immediately before use to convert the fibrinogen to fibrin and activate the factor XIII, thus initiating coagulation of the adhesive.
Commercially available adhesives are of pooled plasma origin. Because blood-derived products have been associated with the transmission of human immunodeficiency virus (HIV), hepatitis virus and other etiologic agents, the acceptance and availability of such adhesives is limited. At present they are not approved for use in the United States.
While the use of autologous plasma reduces the risk of disease transmission, autologous adhesives can only be used in elective surgery when the patient is able to donate the necessary blood in advance.
As noted above, fibrinogen consists of three polypeptide chains, each of which is present in two copies in the assembled molecule. These chains, designated the Aα, Bβ and γ-chains, are coordinately expressed, assembled and secreted by the liver. While it might be expected that recombinant DNA technology could provide an alternative to the isolation of fibrinogen from plasma, this goal has proven to be elusive. The three fibrinogen chains have been individually expressed in E. coli (Lord, DNA 4: 33-38, 1985; Bolyard and Lord, Gene 66: 183-192, 1988; Bolyard and Lord, Blood 73: 1202-1206), but functional fibrinogen has not been produced in a prokaryotic system. Expression of biologically competent fibrinogen in yeast has not been reported. Cultured transfected mammalian cells have been used to express biologically active fibrinogen (Farrell et al., Blood 74: 55a, 1989; Hartwig and Danishefsky, J. Biol. Chem. 266: 6578-6585, 1991; Farrell et al., Biochemistry 30: 9414-9420, 1991), but expression levels have been so low that production of recombinant fibrinogen in commercial quantities is not feasible. Experimental evidence suggests that lower transcription rates in cultured cells as compared to liver may be a factor in the low expression rates achieved to date, but increasing the amount of fibrinogen chain mRNA in transfected BHK cells did not produce corresponding increases in fibrinogen protein secretion (Prunkard and Foster, XIV Congress of the International Society on Thrombosis and Haemostasis, 1993). These latter results suggest that proper assembly and processing of fibrinogen involves tissue-specific mechanisms not present in common laboratory cell lines.
There remains a need in the art for methods of producing large quantities of high quality fibrinogen for use in tissue adhesives and other applications. There is a further need for fibrinogen that is free of blood-borne pathogens. The present invention fulfills these needs and provides other, related advantages.